Amyloidoses represent a spectrum of diseases characterized by the aggregation and deposition of amyloid. Amyloid is a generic term referring to abnormal extracellular and/or intracellular deposits of proteins as fibrils. Amyloid fibrils may be deposited in a variety of vital organs including brain, liver, heart, kidney, pancreas, nerve and other tissues as a consequence of certain inherited and acquired disorders such as Alzheimer's disease, multiple myeloma and related conditions, neuropathies (ATTR amyloidosis), cardiomyopathies, monoclonal plasma cell dyscrasias (AL amyloidosis), adult-onset diabetes, chronic inflammation (AA amyloidosis), aging, bovine spongiform encephalopathy (BSE), Creutzfeld-Jacob disease (CJD), and scrapie.
A. Characterization of Amyloid Proteins
Amyloid is not a uniform deposit and may be composed of unrelated proteins. Proteins that have been identified as capable of forming amyloid in human diseases include immunoglobulin light chains, serum amyloid A protein, β2-microglobulin, transthyretin, cystatin C variant, gelsolin, procalcitonin, PrP protein, amyloid β-protein, ApoA1, and lysozyme. Although these proteins are unrelated, the fibrils which they form have the following common biological properties: 1) they possess a β-pleated sheet secondary structure; 2) they are insoluble aggregates; 3) they exhibit green birefringence after Congo red staining; and 4) they possess a characteristic unbranching fibrillar structure when observed under an electron microscope.
B. Classification of Amyloidosis
Before the major proteins involved in amyloidosis were isolated, sequenced and identified, amyloid was classified based on clinical features into four categories: primary amyloid, secondary amyloid, familial amyloid, and isolated amyloid (U.S. Pat. No. 5,958,883). Primary amyloid is amyloid appearing de novo, without any preceding disorder. Secondary amyloid appears as complication of a previously existing disorder. Patients with rheumatoid arthritis, osteoarthritis, or ankylosing spondylitis can develop secondary amyloidosis as do patients with tuberculosis, lung abscesses, and osteomyelitis.
Familial form of amyloid is found in patients diagnosed with genetically inherited forms of amyloid. Several geographic populations have been identified with such forms of amyloid. One form of inherited amyloid is found in patients with Familial Mediterranean Fever. Sephardic Jews in Israel possess a genetic predisposition for Familial Mediterranean Fever. A second form is found in patients diagnosed with Familial Amyloidotic Polyneuropathy. Patients of three different nationalities, Swedish, Portuguese, and Japanese have been shown to possess a genetic predisposition referred to as Familial Amyloidotic Polyneuropathy.
Unlike the other three forms of amyloid, isolated amyloid only tends to involve a single organ system. Type II diabetic patients have isolated amyloid deposits in the pancreas restricted to the beta cells in the islets of Langerhans. A serious complication of long-term hemodialysis is amyloid deposited in the medial nerve and clinically associated with carpal tunnel syndrome. In Alzheimer's disease, amyloid deposition is restricted to the central nervous system (CNS). Similarly, in Down's syndrome patients, deposition of amyloid occurs in the brain when the patient reaches approximately 35 years of age.
Currently, amyloid is classified according to major protein type found associated with the disorder (U.S. Pat. No. 5,958,883). The major proteins isolated and sequenced are AA amyloid, AL amyloid, transthyretin, β2-microglobulin, procalcitonin, β amyloid protein or β/A4, and Prp protein.
AA amyloid is commonly found in a host of seemingly unrelated disorders including chronic inflammation, various forms of malignancy, and in Familial Mediterranean Fever. AA amyloid is also found in animal models which use daily repeated injections of pro-inflammatory stimulants such as casein or azocasein. In these animals, amyloid deposition is detected in the spleen, liver, and kidney within 7-10 days of the initial injections. The isolated AA amyloid protein is approximately 76 amino acids long and has a molecular weight of about 85 Kda. These 76 amino acids correspond to the amino terminal ⅔s of the naturally occurring serum protein, serum amyloid A (SAA). SAA is an acute phase protein whose concentration increases by a thousand-fold within 24 hours during any inflammatory disorder. It is made by the liver, but current research has suggested that it is present in other tissues as well.
AL amyloid usually occurs secondary to multiple myeloma, or B-cell type malignancies, and other plasma cell dyscrasias. Not all patients with multiple myeloma develop AL amyloid; only 10 to 15% develop AL amyloid related clinical problems. AL amyloid is usually due to the deposition of the variable region of immunoglobulin light chains, either lambda or kappa chain, but the entire light chain may also be present. Due to the inherent amino acid sequence diversity seen in the variable region of the light chains, AL amyloid isolated from different patients differs in its amino acid sequence. However, within a single patient the sequence of the AL amyloid protein is constant regardless of the organ from which the amyloid is isolated.
Transthyretin (TTR) is another name for prealbumin. Prealbumin or TTR is the serum carrier of thyroxine, retinol binding protein, and retinoic acid. It is synthesized by the liver and consists of 127 amino acids. TTR forms amyloid deposits in patients with certain familial amyloid polyneuropathy (FAP). It has been determined that TTR found in the deposits has various amino acid substitutions compared to circulating transthyretin of normal individuals. Single amino acid substitutions have been identified at a number of positions within the molecule, including: 30, 33, 60, 77, 84, 111, and 122. These were identified in TTR molecules isolated from amyloid deposit of FAP patients. The most common substitution is a methionine for valine at position 30 of the molecule.
β2-Microglobulin is present in the amyloid deposits of patients having serious complications from long-term hemodialysis. It is also associated with disorders such as carpal tunnel syndrome, joint swelling, multiple spontaneous fractures, and radiolucency in the wrist and hip. β2-Microglobulin is a single polypeptide chain of 100 amino acid residues and has a molecular weight of about 11.8 Kda. It accumulates not only in the blood of uremic patients but also in the synovial fluid and in the tissues.
Procalcitonin is found in the amyloid deposits of endocrine tumors which secrete calcitonin. Medullary carcinoma of the thyroid is an example of such a tumor. The tumor is related to the C-type cells of the thyroid which normally secrete calcitonin.
β-Amyloid protein or β/A4 is deposited in the brains of patients with Alzheimer's disease as well as Down's syndrome patients over the age of 35. β-Amyloid protein has a molecular weight of 4.2 Kda and is derived from a larger precursor molecule. The β-amyloid precursor molecule may take on different forms, including proteins of 695, 714, 751, and 770 amino acids, since the precursor gene produces at least four principle mRNAs through alternative splicing of two exons.
PrP protein, also known as the prion protein PrP 27-30, has a molecular weight of 27 to 30 Kda and is derived from a larger protein, PrP Sc. These proteins are highly infectious and transmissible. They are present in the amyloid deposits in neurological disease such as Creutzfeldt-Jakob disease, Gerstmann Strausiler Syndrome, Scrapie, Bovine Spongiform Encephalopathy, and Kuru. These diseases are rapidly progressive neurological disorders characterized by dementia and fall into the category of subacute spongiform encephalopathies. Microscopically, the cerebral tissue is characterized by neuronal loss, gliosis, spongiform changes, and extracellular amyloid deposits in the form of plaques.
Although there exist many biochemically diverse types of amyloid deposits, the pathogenetic mechanisms that may be operating in amyloidosis in general are shared by the different types. In most cases, a circulating precursor protein may result from overproduction of either intact or aberrant molecules (plasma cell dyscrasias), reduced degradation or excretion (SAA in some secondary amyloid syndromes and β2-microglobulin in long-term hemodialysis) or genetic abnormalities associated with variant proteins (FAP). Proteolysis of a larger precursor molecule occurs in many types of amyloidosis, resulting in the production of lower MW fragments that polymerize assume a β-pleated sheet conformation and deposit as fibrils in tissues, usually in an extracellular location. Amyloid deposition is generally an irreversible pathologic process. In most instances, the constant accumulation of amyloid fibril proteins leads to progressive organ dysfunction and eventual death.
C. Animal Models for Amyloidosis
Elucidation of the pathogenesis, treatment and prevention of diseases associated with amyloid fibril deposition has been hampered by the lack of suitable in vivo experimental models that can reproduce salient features of amyloidosis. One approach that has been extensively used to study AA amyloidosis involves injecting mice with one of a variety of chemical or biological compounds including casein, silver nitrate, and lipopolysaccharide (Skinner et al., 1997; Kisilevsky et al., 1994). These agents stimulate the production of cytokines, e.g., tumor necrosis factor and interleukins 1 and 6 (IL-1 and IL-6), that mediate the inflammatory response by increasing the synthesis of serum amyloid A protein (SAA) (Sipe et al, 1994). This molecule, in turn, serves as the precursor of the polypeptide that is deposited in the spleen, liver, and kidneys of affected animals as Congophilic, green birefringent fibrils, i.e., amyloid. Amyloid deposition can be accelerated in mice injected intravenously with amyloid enhancing factor (AEF), a molecule isolated from spleens of mice with AA amyloidosis (Axelrad et al., 1982). The advantage of this model is the reproducibility of the pathologic manifestations and the predictable sites of deposition. Amyloid invariably develops within the liver, spleen, and kidney (Husebekk et al. 1985; Tape et al., 1988; Sipe et al., 1992; Husby et al., 1994). However, because cessation of the inflammatory stimulus results in disappearance of the amyloid deposits (Kisilevsky et al., 1994), the usefulness of this model is limited by the need for repeated injections and the transitory nature of the induced pathology.
Through transgenic technology, it is now possible to study the pathological effects of continuous expression of cytokines and other biological factors. Since IL-6 plays a seminal role in hematopoiesis and the inflammatory-mediated response (Kishimoto et al., 1988; Li et al., 1989; Hirano et al., 1990; Van Snick et al., 1990; Fattori et al., 1994), transgenic mice expressing the murine (mIL-6; Brandt et al., 1990; Woodroofe et al., 1992) or human (hIL-6; Suematsu et al., 1989; Fattori et al, 1989) form of this molecule have been generated. Animals carrying the hIL-6 gene under the control of either the human Eμ enhancer (Suematsu et al., 1989) or the mouse metallothionein-1 (MT-1) promoter (Fattori et al., 1989) predominantly express IL-6 in B cells or liver, respectively. The Eμ/hIL-6 transgenic mice are typified by an extensive polyclonal plasma cell proliferation within lymph nodes and spleen, as well as mesangioproliferative glomerulonephritis (Suematsu et al., 1989). The MT-1/hIL-6 animals have a sustained increase in liver-derived acute phase proteins and an IgG plasmacytosis within lymphoid tissue; in addition, these mice manifest renal pathology resembling that seen in patients with myeloma (cast) nephropathy (Fattori et al., 1989). Furthermore, systemic AA-amyloid deposition is detected in these animals when they reach 3 months of age. The extent of the amyloid deposition increases steadily with age and occurs primary in the spleen, liver, and kidneys (Solomon et al., 1999), although the heart, adrenal glands and pancreas may also be involved.
D. Methods of Treatment for Amyloidosis
Very rarely do patients with clinically proven amyloidosis spontaneously achieve complete remission, probably because the amyloid fibrils themselves are non-immunogenic. Various therapies for amyloidosis have been investigated, such as high-dose chemotherapy, steroids, iodinated doxorubicin, and stem cell replacement therapy. However, in only one type of amyloid disease, Familial-Mediterranean amyloidosis, has drug treatment (with colchicine) been shown to be effective. To date there is no treatment for Alzheimer's disease at any stage of its development. Two therapeutic reagents, Cognex and Menthane, appear to give slight relief to some victims but do not alter the course of the disease. Since Alzheimer's disease and related degenerative brain disorders are a major medical issue for an aging population, the need for new treatments and methods for diagnosing the disorders are needed. Thus, therapeutic efforts in amyloidosis have been focused on the development of compounds and agents that prevent protein aggregation leading to the formation of fibrils, as well as agents that block the initial interaction of fibrillar proteins in tissues.
A variety of studies have characterized antibodies that bind to amyloid proteins or amyloid fibrils. (See, for example, U.S. Pat. Nos. 5,714,471; 5,693,478; 5,688,651; 5,652,092; 5,593,846; 5,536,640; 5,385,915; 5,348,963; 5,270,165; 5,262,332; 5,262,303; 5,164,295; and 4,782,014.) In addition, several publications have suggested that anti-amyloid antibodies might be useful for studying the progression of beta-amyloidosis and for various therapeutic options. (See, for example, Bellotti et al., 1992; Bellotti et al., 1993; Walker et al., 1994; and Bickel et al., 1994)
U.S. Pat. No. 6,017913 teaches the use of naphthylazo compounds to inhibit amyloid aggregation in a mammal. U.S. Pat. No. 5,744,368 discloses methods and compositions for preventing aggregation of amyloid β-protein (β-AP) comprising providing a β-AP binding compound, such as transthyretin, to promote complex formation between β-AP and β-AP binding protein and prevent β-AP from self-aggregating and forming amyloid.
E. Diagnosis and Classification of Amyloid Proteins from Microscopic Slides by Analytical Procedures
The diagnosis of amyloid deposition is almost exclusively done by the morphologic examination of tissue material taken from biopsy samples or autopsied organs. Positive staining with Congo red and exhibition of green birefringence in polarized light are generally accepted principles, which suggest the deposition of amyloid in the examined sample. The finding of fibrils by electron microscopy in biopsies or tissues under examination confirms the diagnosis of amyloidosis. Ongoing research in the last decades has revealed that different proteins can be deposited as amyloid fibrils. At least 18 different proteins have been discovered as constituents of amyloid so far. It is of decisive importance for the prognosis and therapy of amyloid related diseases to discover the very nature of the proteins it the deposits. Immunohistochemical methods are mostly employed to specify which protein constitutes the fibrils. But it has been frequently reported, that some antibodies used in this techniques failed to react or give only weak reactions with the proteins entwined in the amyloid fibrils though they bind specifically to the precursor molecules. Other methodical approaches have to be taken to get an unequivocal definition of the proteins, which are deposited as amyloid fibrils. Another way to reach this goal is to isolate the fibril proteins from the tissue and perform an amino acid sequence analysis of the purified protein. This procedure poses no problems in cases of systemic amyloidosis, where after an autopsy enough fresh tissue material is available for extraction. But in many cases only microscopic slides or paraffin embedded tissue blocks are available for further examination. Normally tissue samples are formalinized and dehydrated before they are embedded in paraffin. There are a few reports about sequencing formalinized material and deriving useful information about the sequence of the amyloid related protein in the sample examined.